Bacteria have evolved strategies to resist host defense mechanisms including resistance to antimicrobial peptides (APs). The sap (sensitivity to antimicrobial peptides) operon plays a significant role in this defense, as demonstrated by attenuation of sap operon mutants in animal models. The hypothesis of this proposal is that the sap operon plays a significant role in nontypeable Haemophilus influenzae (NTHI) survival in a chinchilla model of otitis media by maintaining resistance to APs in vivo and by up-regulating expression of specific AP resistance determinants. Expression of the sap operon during the disease course of otitis media or when exposed in vitro to chinchilla neutrophils or neutrophil granule extract will be analyzed by flow cytometry and histology. Mutations in the six genes of the NTHI sap operon will define the functional requirement for each gene in vivo. Finally, microarray analysis will be used to identify genes that determine resistance to APs as a result of sap operon expression. The data generated from these studies will significantly advance the field in understanding how NTHI adapt to the innate immune response in vivo in microenvironments encountered during disease progression and will identify novel virulence mechanisms resulting from sap operon expression that enable NTHI to survive and cause disease.